人基质金属蛋白酶3基因RNA慢病毒载体构建及在人退变髓核细胞中的鉴定

doi:10.3969/j.issn.2095-4344.2016.07.005

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (7): 947-956.doi: 10.3969/j.issn.2095-4344.2016.07.005

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇下一篇

人基质金属蛋白酶3基因RNA慢病毒载体构建及在人退变髓核细胞中的鉴定

曹进，傅培荣，房晶，杨建坤，位华卫，李思源，高峰，西永明

青岛大学附属医院脊柱外科，山东省青岛市 266003

收稿日期:2015-11-24 出版日期:2016-02-12 发布日期:2016-02-12
通讯作者: 西永明，博士，副教授，主任医师，青岛大学附属医院脊柱外科，山东省青岛市 266000
作者简介:曹进，男，1985年生，河南省周口市人，汉族，青岛大学在读硕士，主要从事骨科的基础和临床研究。
基金资助:
国家自然科学基金(81470104)

Constructing and identifying a lentiviral vector of RNA interference targeting matrix metalloproteinases-3 gene in human degenerative nucleus pulposus cells

Cao Jin, Fu Pei-rong, Fang Jing, Yang Jian-kun, Wei Hua-wei, Li Si-yuan, Gao Feng, Xi Yong-ming

Department of Spine Surgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China

Received:2015-11-24 Online:2016-02-12 Published:2016-02-12
Contact: Xi Yong-ming, M.D., Associate professor, Chief physician, Department of Spine Surgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
About author:Cao Jin, Studying for master’s degree, Department of Spine Surgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
Supported by:
the National Natural Science Foundation of China, No. 81470104

摘要/Abstract

摘要：

文章快速阅读：

文题释义：

基质金属蛋白酶3：属基质金属酶类家族成员之一，具有细胞外基质降解作用，相对分子质量54 000，可消化基底膜中的Ⅳ型胶原，并能激活基质金属蛋白酶1前体使之形成基质金属蛋白酶1，可降解细胞外基质中的胶原纤维和明胶，从而有利于肿瘤的侵袭和转移。

髓核：是位于软骨板和纤维环中间，由纵横交错的纤维网状结构即软骨细胞和蛋白多糖黏液样基质构成的弹性胶冻物质。髓核在出生时体积大而松散，位于椎间盘的中央，至成年时位置移至椎间盘的中后部。在20岁以前构成髓核的主要物质是大量蛋白多糖复合体、胶原纤维和纤维软骨，随着年龄的增长，髓核中的蛋白多糖解聚增多，水分逐渐减少，胶原增粗并逐渐被纤维软骨所替代。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

ORCID:0000-0002-2921-0699(Xi Yong-ming)

Abstract:

BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracellular matrix components such as type II collagen and aggrecan.
OBJECTIVE: To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells.
METHODS: According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI and EcoRI enzymes, and then transfected into the competent cells. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cells were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours.
RESULTS AND CONCLUSION: The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfully constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

曹进，傅培荣，房晶，杨建坤，位华卫，李思源，高峰，西永明. 人基质金属蛋白酶3基因RNA慢病毒载体构建及在人退变髓核细胞中的鉴定[J]. 中国组织工程研究, 2016, 20(7): 947-956.

Cao Jin, Fu Pei-rong, Fang Jing, Yang Jian-kun, Wei Hua-wei, Li Si-yuan, Gao Feng, Xi Yong-ming. Constructing and identifying a lentiviral vector of RNA interference targeting matrix metalloproteinases-3 gene in human degenerative nucleus pulposus cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(7): 947-956.

图/表（结果） 6

PCR identification of lentiviral vectors

LV3-H1-GFP/puro was used as expression vector. The recombinant LV3-MMP3-shRNA was digested by XbaI and NheI. Then the positive clones were identified by PCR using agarose gel electrophoresis, as shown in Figure 1. The size of the positive clone was 8 000 bp.

DNA sequencing results

The positive clone was analyzed by sequencing. The result of the sequence identification showed the sequences were consistent with the design, as shown in Figure 2. After sequences BLAST in Genebank, it was confirmed that the ds-oligo DNA sequences were successfully inserted into the expression lentiviral vector pGLV3.

The determination of virus titer

The four plasmids of the lentiviral vector: pGLV3-shMMP3, pGag/Pol, pRev and pVSV-G were co-transfected to 293T cells to package lentiviral particles. After 72 hours, the supernatant was collected and the titer of virus was measured by detecting the expression level of EGFP (Figure 3). The recombinant lentivirus was harvested from 293T cells with a viral titer of 1-5×10⁸TU/mL.

Transfection efficiency and lentiviral identification

After transfection with lentiviral-EGFP, degeneration IVD nucleus cells showed green fluorescence under a microscope at 72 and 96 hours (Figures 4 and 5). The transfection efficiency was high, and the positive cells accounted for 90% of the total cells. When the multiplicity of infection=100, the transfection efficiency of experimental group and negative control group reached 90%, as shown in Figure 4.

Effect of recombinant plasmids on MMP3 mRNA

Reaction products were examined with 1% agarose gel electrophoresis. The results of RT-PCR showed that the product size was consistent with that the expected value (Table 2), and there was no specific amplification as shown in Figure 5. After transfection for 72 and 96 hours, the expression of MMP3 mRNA in the 6076 transfection group was significantly different from that in the blank control group and LV3NC group (P < 0.05). The comparative ??CT method was used to evaluate the gene expression of MMP3. RNAi targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively (Table 3).

Effect of recombinant plasmids on MMP3 protein expression levels

After transfection for 72 hours, the expression of MMP3 protein in the four experimental groups was higher than that in the blank control group and LV3NC group. However, the expression of MMP3 in the four experiment groups was lower than that in the blank control group and LV3NC group at 96 hours after transfection. More importantly, the expression of MMP3 in the 6076 group was significantly lower than that in the others groups (Figure 6), and at 96 hours, the interfering efficiency of protein expression was 57.2%.

参考文献

[1] Kadow T, Sowa G, Vo N, et al. Molecular basis of intervertebral disc degeneration and herniations: what are the important translational questions? Clin Orthop Relat Res. 2015;473(6):1903-1912.

[2] Hoy D, Bain C, Williams G, et al. A systematic review of the global prevalence of low back pain. Arthritis Rheum. 2012;64(6):2028-2037.

[3] Weber KT, Jacobsen TD, Maidhof R, et al. Developments in intervertebral disc disease research: pathophysiology, mechanobiology, and therapeutics. Curr Rev Musculoskelet Med. 2015;8(1):18-31.

[4] Risbud MV, Shapiro IM. Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol. 2014;10(1):44-56.

[5] Roberts S, Caterson B, Menage J, et al. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine (Phila Pa 1976). 2000;25(23):3005-3013.

[6] Weiler C, Nerlich AG, Zipperer J, et al. 2002 SSE Award Competition in Basic Science: expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption. Eur Spine J. 2002;11(4):308-320.

[7] Le Maitre CL, Freemont AJ, Hoyland JA. Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc. J Pathol. 2004; 204(1):47-54.

[8] Wang X, Li F, Fan C, et al. Effects and relationship of ERK1 and ERK2 in interleukin-1β-induced alterations in MMP3, MMP13, type II collagen and aggrecan expression in human chondrocytes. Int J Mol Med. 2011;27(4):583-589.

[9] Murphy G, Cockett MI, Stephens PE, et al. Stromelysin is an activator of procollagenase. A study with natural and recombinant enzymes. Biochem J. 1987;248(1): 265-268.

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[12] Yurube T, Takada T, Suzuki T, et al. Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration. Arthritis Res Ther. 2012;14(2):R51.

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引言

Lumbar intervertebral disc (IVD) degeneration (IVDD) is a major cause of low back pain^[1], which is well documented as a global health concern^[2], not only because of its high morbidity, but also because of its high socioeconomic costs and poor therapeutic effects. Current evidence implicates the predominant changes to the IVD are characterized by active cell number reduction, extracellular matrix (ECM) degradation, altered phenotype of normal disc cells, and presence of inflammation^[3-4]. Excessive destruction of the ECM, especially loss of collagen type II and aggrecan, plays a critical role in promoting the occurrence and development of IVDD.

Matrix metalloproteinases (MMPs) play a dominant role in IVD matrix degradation^[5-7]. Among them, MMP3 (stromelysin-1) is considered as a key enzyme involved in direct degradation of ECM components such as collagen type II and aggrecan^[8] and indirect ECM degradation by activating other latent MMPs^[9]. Increasing evidences have shown that MMP3 isup-regulated in human degenerative lumbar nucleus pulposus tissues and in an animal disc degeneration model compared to normal nucleus pulposus tissues^[10-15]. Therefore, to control the

proteolytic activity of MMP3 gene by an efficient technique will provide an effective prevention or therapeutic strategy against development or progression of lumbar IVDD when aiming to develop new treatment options for early disc degeneration.

RNA interference (RNAi) is an endogenous process that maintains the homeostasis by regulating gene expression. The technology of RNAi that uses small interfering RNA (siRNA) is an effective method that is used to silence the genes^[16-18]. The silencing of MMP3 by siRNA has been reported in tumor cells^[19-20]. Till now, there are no studies addressing the effects of MMP3 silencing in human degenerative IVD cells by RNAi.

The present study attempted to construct the short hairpin RNA (shRNA) lentiviral vectors targeting human MMP3 gene and to detect the efficiency of gene silence by infecting human degenerative nucleus pulposus cells.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

材料方法

Design

A genetic study.

Time and setting

Experiments were performed from June 2015 to October 2015 in the Central Laboratory of the Affiliated Hospital of Qingdao University, Qingdao, China.

Materials

Competent cells and PGLV3 were purchased from Shanghai GenePharma Co., Ltd., China and 239T cells were reserved in the Center Laboratory of the Affiliated Hospital of Qingdao University, China.

The degenerative human nucleus pulposus samples were donated from patients (15 cases; mean age, 26 years; range, 20-32 years; 9 males and 6 females) with IVDD during IVD discectomy, who had no clinical history of other diseases. All patients provided informed consent according to the latest version of the Helsinki Declaration. The study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University, China. Routine MRI scan of the spine was performed for all patients diagnosed as having IVDD. The severity of disc degeneration was graded I-III according to the Pfirrmann classification^[21].

Methods

Design of oligonucleotides (oligo) targeting MMP3 gene

As shown in Table 1, four pairs of MMP3 shRNA were designed with Designer3.0 software (GenePharma, Shanghai, China) according to the human MMP3 mRNPA (NM_002422.4) sequence obtained from GenBank (http://www.ncbi.nlm.nih. gov/genbank) and the shRNA design principles. Double-stranded DNA fragments, with cohesive termini of the EcoRI and BamHI restriction enzymes (MBI Fermentas) and hairpin sequence, were synthesized in vitro.

Annealing of DNA oligo sequences targeting MMP3 gene

The sense strand and antisense strand DNA oligo solutions from DNA oligo (100 μmol/L) dissolved TE (pH 8.0) were configured according to the following proportions annealing reaction system: 10×shDNA annealing buffer 5 μL, sense strand (100 μmol/L) 5 μL, antisense strand (100 μmol/L) 5 μL, ddH₂O 35 μL, total 50 μL. The target sequence was amplified by PCR under: 95 ℃ for 5 minutes; 85 ℃ for 5 minutes; 75 ℃ for 5 minutes; 70 ℃ for 5 minutes. The double-stranded DNA fragments (10 μmol/L) gained from the above process were inserted into LV3 by diluting 50 times.

Construction of LV3-shRNA vectors

LV3 vectors (10 μg) were digested by BamHI and EcoRI enzyme at a 37 ℃ for 1 hour according to the following system: 2×Buffer Tango 10 μL, BamHI 5 μL, EcoRI 5 μL, LV3 10 μL, ddH₂O to 100 μL, total 100 μL. The concentration of Reaction products estimated by agarose gel electrophoresis and the linearized pGLV3 lentiviral vectors was diluted to 50 ng/μL.

The double-stranded DNA fragments were ligated into the BamHI and EcoRI-digested site of expression vector pGLV3 at a 22 ℃ for 1 hour according to the following system: 10×T4 ligation buffer 2 μL, LV3 (BamHI+EcoRI) 1 μL, shDNA template (100 nmol/L) 1 μL, T4 DNA ligase (5 weissU/μL, MBI Fermentas) 1μL, ddH₂O 15 μL, total 20 μL. E. coli DH5α was grown in LB medium containing 50 mg/L ampicillin and cultured at 37 ℃ for 16 hours. The recombinant lentiviral vector expressing specific shRNA of MMP3 gene (10 µL) was added into the competent cells prepared by calcium chloride.

Identification and sequencing of positive clones

Four colonies picked up from the plate cultured well were placed in LB medium tube containing 5 mL ampicillin (50 µg/mL). These tubes were placed in a shaker for 16 hours under the following condition:

37 ℃, 250 r/min. The plasmids were extracted from the above liquid following the instruction of TIANprep Rapid Mini Plasmid Kit (Hangzhou Axygen Biological Technology Co., Ltd., China). The recombinant LV3-MMP3-shRNA was confirmed by restriction enzyme digestion by XbaI and NheI. The correct sample was sent to Shanghai GenePharma Company, China for the sequence identification. Bacterial strains without errors were extracted by high pure plasmid extraction kits.

Packaging of lentiviral

The shuttle plasmids containing target sequence and packaging plasmids (pGag/Pol, pRev, pVSV-G) were placed in 5 mL sterile centrifuge tube containing 1.5 mL serum-free DMEM medium (Gibco, USA). 300 μL RNAi-Mate was added in another tube containing 1.5 mL serum-free DMEM medium. After 5 minutes placed at room temperature, the liquids in the two tubes were mixed together and placed at room temperature for 20-25 minutes. The mixture was added in 15 mL culture dish containing 293T cells and incubated in a 37 ℃ humidified 5% CO₂ and 21% O₂ incubator for 4-6 hours. Then the transfer liquid were discarded and added in DMEM/F12 with 18% fetal bovine serum (FBS) (Gibco) and incubated in a 37℃ humidified 5% CO₂ and 21% O₂ incubator. Lentiviral were collected from supernatants by conventional methods.

Detection of lentiviral titer

The 293T cells in 96-hole plates at a density of 3×10⁴ cells per well were incubated in a 37℃ humidified 5% CO₂ and 21% O₂ incubator for 24 hours. 10 μL lentivirus liquid was diluted to four gradients by DMEM containing 10% FBS 10 times. At the same time, a blank control group was established. After 24 hours, 100 μL diluted virus needed determination instead of culture medium was added into each group and incubated in the 37 ℃ humidified 5% CO₂ and 21% O₂ incubator. After 72 hours, the supernatant was collected and the titer of virus was measured by detecting the expression level of enhanced green fluorescent protein (EGFP).

Cultivation of human degenerative nucleus pulposus cells

Fresh nucleus pulposus tissue harvested was immediately washed twice in PBS to remove blood cells. The surrounding AF was carefully removed, and the nucleus pulposus was cut into pieces, approximately 1 mm³ in size. The matrix was digested 20 minutes at 37 ℃ in 0.25% trypsin solution, following 3-4 hours in 0.2 % type II collagenase. The enzymatically digested cells were filtered through 100-μm filter and suspended in DMEM/F12 (1:1) supplemented with 15% FBS and 1% penicillin/streptomycin at 37 ℃ in a 5% CO₂ humidified incubator. The culture medium was changed every 2 days. When the cells reached 90% confluence after 10 days, they were treated with a 0.25% trypsin solution and subcultured in a 25-mL culture flask (1×10⁵/mL, 5 mL per flask). All experiments were carried out with human nucleus pulposus cells at passages 2-3.

Cell grouping and gene transfection

Passage 2 human degenerative nucleus pulposus cells were digested with 2.5 g/L trypsin, counted, and plated in 6-well culture plates at a density of 1×10⁵ cells per well containing DMEM/F12 (1:1) supplemented with 15% FBS and 1% penicillin/streptomycin at 37 ℃ in a 5% CO₂ humidified incubator. Cells were divided into six groups: the four groups of human degenerative nucleus pulposus cells transfected by lentivirus respectively carrying 6072, 6074, 6076 and 6077 (experimental group, named 6072, 6074, 6076 and 6077 groups), the group of human degenerative nucleus pulposus cells transfected by empty lentivirus (negative control group, LV3NC group), and the group of human degenerative nucleus pulposus cells with no treatment (blank control group). After 24 hours, the culture medium was removed. Lentivirus liquid at 1×10⁷ virus titer was added into groups as previously described, respectively. Culture medium instead of the virus served as blank control group. After 24 hours transferred by the virus, lentivirus liquid was replaced by original culture medium. The cells always were maintained in the 37 ℃, humidified 5% CO₂ and 21% O₂ incubator. At 72, 96 hours after transfection, the cells were observed by inverted fluorescence microscope (Nikon, Japan) and were collected in each group. The transfection efficiency of each group was calculated by inverted fluorescence microscope.

Determination of MMP3 mRNA expression by reverse transcription (RT)-PCR

At 72 and 96 hours after transfection, cells were fully cracked by Trizol reagent, and chloroform was added into the supernatants. Blended by concussion, the supernatants were added isopropyl alcohol and centrifuged, and then the mRNA was diluted with ultrapure water after being washed with 75% ethanol. Absorbance (A) value and concentration of the RNA were measured with the spectrophotometer, and the purity was detected by RNA electrophoresis. Total RNA was extracted and cDNA was synthesized with RT-PCR following the reaction conditions: 42 ℃ 30 minutes, 85 ℃ 10 minutes. The RT reaction conditions: 2×RT buffer 10 μL, RT primer (1 μmol/L) 1.2 μL, total RNA 2 μL, MMLV reverse transcriptase (200 U/μL) 0.2 μL, DEPC water 6.6 μL, total 20 μL. Then real-time fluorescence quantitative PCR was carried out with RTFQ PCR kit following the instruction. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 12 seconds, annealing at 62 ℃ for 40 seconds, total 40 cycles. Then the mixtures were denatured at 95 ℃ for 60 seconds at the end of the PCR process, and cooled to 55 ℃. Sequences of the primer refer to Table 2. Reaction products were examined with 1% agarose gel electrophoresis. A values of the electrophoresis bands were analyzed with gel imaging system to verify the stripes of target gene. Quantitative analysis was performed using the ratio of the target gene to hACTB. The PCR reaction conditions: 2× quantitative PCR Master Mix 10.0 μL, primer F (20 μmol/L) 0.08 μL, primer R (20 μmol/L) 0.08 μL, cDNA 2 μL, Taq DNA polymerase (2.5 U/μL) 0.4 μL, ddH₂O 7.44 μL, total 20 μL.

Western blot analysis of MMP3 expression

Western blot was used to test the protein expression of MMP3 in human degenerative nucleus pulposus cells. The cells from each group were harvested at 72 and 96 hours after transfection and cell lysate solution (100 μL) was added. 10 μL supernatant was incubated in 2×SDS- PAGE loading buffer at 100 ℃ for 5 minutes and cooled on the ice, then to centrifuge (12 000 r/min at 4 ℃) for 10 minutes. Proteins were separated on 10% SDS-PAGE, and then transferred to polyving lidene fluoride membranes, followed by blocking for 2 hours. The membranes were incubated with primary antibodies (diluted 1:300, Abcam, UK) at 4 ℃ overnight, rinsed three times with PBS-T (15 minutes once) on the second day, and subsequently incubated with secondary antibodies (labeled by horseradish peroxidase, 1:2 000) at 37 ℃ for 2 hours. Then the membranes were washed with PBS-T for three times (15 minutes/time), and followed by rinsing three times with PBS for 15 minutes. Finally, the membranes were colored in SuperSignal West Pico Chemiluminent Substrates (PIERCE), then developed, fixed and exposed in the cassette. The software Gel-Pro Analyzer (Media.cybernetics.l.p.) was used to measure A value of every zonel.

Main outcome measures

Real time-PCR and western blot were used to identify the interfering efficiency of each group at 72 and 96 hours.

Statistical analysis

All data were collected as mean and analyzed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Intergroup comparison was done using the Student’s t-test for two groups, or analysis of variance for multiple groups. A P value less than 0.05 was considered statistically significant.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

讨论

Currently, all available treatments for IVDD are palliative and can do nothing to slow down or reverse the course of IVDD. Gene therapy, an efficient preventive or therapeutic strategy against development or progression of IVDD, is usually defined as the use of nucleic acid transfer, either RNA or DNA, to treat or prevent a disease. As for a successful gene therapy, one of technical issues that need to be considered is usually genetic materials that are not well received by cells; therefore, vectors are required to deliver the gene of interest into the target cells^[22].

RNAi is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. siRNAs, introduced into cells and produced via endonucleolytic processing by the ribonuclease Dicer of exogenously introduced long, double-stranded RNA, are transferred to Argonaute proteins, which guide the sequence-specific silencing of homologous target messenger RNAs that contain complementary sequences by either enzymatically cleaving the mRNA or repressing its translation^[23]. The therapeutic application of siRNA is impressively promising due to efficacy, target specificity and preclinical safety, as demonstrated in selected in vitro and in vivo studies^[24].

However, delivery of genetic materials to their target cells is the biggest obstacle faced by siRNA therapies before they can exert their gene silencing activity. Viral vectors and non-viral vectors, two different types of vectors, have been found. Because of its efficiency, viruses are have been used in numerous gene therapy studies on IVDD^[25].

Compared with non-viral vectors, viruses are very efficient vectors because their entry into cells and the subsequent expression of their genes are part of the natural viral life cycle. With the continuous improvement of genetically modified HIVs, these early marker genes carrying HIVs have been evolved into safe and effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles^[26], including (1) capacity of delivery large gene segment; (2) non-toxicity, not prone to induce the host immune response, good security and high transfection efficiency; (3) the capability of infecting not only the dividing cells but also terminally differentiated cells and non-dividing cells. It is one of the most effective carriers for transferring the target gene in gene therapy and RNAi. In this study, we used lentiviral vectors and obtained very high transfection efficiency. The observation results are consistent with the previous studies^[27-29]. Safety concerns of lentiviral vectors are receiving more attentions. Expression vector LV3-H1-GFP/puro and packaging plasmids (pGag/Pol, pRev, pVSV-G) we used were safe because the HIV-1 encoding gene was deleted and transformed to self-inactivating,

In this study, by using target sequences analysis of software Designer3.0 and sequences BLAST in Genebank, four interference points targeting human MMP3 gene were designed and four complementary DNA sequences with cohesive termini of the EcoRI and BamHI restriction enzymes were synthesized in vitro. In addition, the ds-oligo DNA which expressed MMP3 shRNA was inserted into the linearized pGLV3 lentiviral vectors digested by BamHI and EcoRI enzyme. The recombinant H1/GFP&Puro-MMP3-shRNA was constructed and confirmed by restriction enzyme digestion by XbaI and NheI by electrophoresis and sequencing. The analysis using restriction endonuclease digestion and sequencing confirmed that the ds-oligo DNA was successfully inserted into the lentiviral vector. Accordingly, the correct interference vector targeting human MMP3 gene was obtained.

Then, the four plasmids of the lentiviral vectors: LV3 (H1/GFP&Puro)-MMP3, pGag/Pol, pRev and pVSV-G were co-transfected to 293T cells by RNAi-Mate to package lentiviral particles. After 72 hours, the supernatant was collected and the titer of virus was measured by detecting expression level of EGFP. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1-5×10⁸ TU/mL.

Human degenerative nucleus pulposus cells were infected with LV3 (H1/GFP&Puro)-MMP3 shRNA, and the infection rate over 90% detected by EGFP under inverted fluorescence microscope, which showed that lentivirus containing RNAi targeting MMP3 gene had been successfully transfected to human degenerative nucleus pulposus cells.

Human degenerative nucleus pulposus cells were infected by lentivirus, and the interfering efficiency was detected by RT-PCR and western blot. The plasmid with the highest interfering efficiency was the group contained GCC AGG CTT TCC CAA GCA AAT at 72 hours after transfection and the interfering efficiency respectively was 98%. Unexpectedly, the expression of MMP3 protein was not declined but higher than the others at 72 hours. The suppression of MMP3 mRNA was not consistent with that of MMP3 protein in the group targeting 6076 sequence at 72 hours, accounting for compensatory adjustment function of the cell itself or protein turnover rate. RNAi targeting the 6076 sequences in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively, which may has been caused by the transcriptional activity or the transcriptional activity of cells.

The recombinant lentiviral vector was successfully constructed in this study, which contained the RNAi targeting human MMP3 gene. It was interfered successfully at the cellular level. Our experimental findings lay a foundation for the subsequent study of the function and gene therapy of MMP3 gene in human degenerative nucleus pulposus cells.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

人基质金属蛋白酶3基因RNA慢病毒载体构建及在人退变髓核细胞中的鉴定

Constructing and identifying a lentiviral vector of RNA interference targeting matrix metalloproteinases-3 gene in human degenerative nucleus pulposus cells

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